Basic Parts
| ID | Part Name | Short Description |
|---|---|---|
| BBa_K4586000 | CCP1 | This part codes for cyclic citrullinated peptide which is a synthetic peptide designed to detect and measure specific antibodies that are elevated in autoimmune diseases, especially rheumatoid arthritis. |
| BBa_K4586001 | Mouse Notch Core | This part codes for an enhanced mouse notch core, which is a notch-derived transmembrane regulatory protein that normally regulates cell fate decisions during a variety of developmental processes. |
| BBa_K4586002 | ZF21.16 | Zinc finger proteins are considered one of the most unique and huge collections of proteins that are variable in their biological function and structure. |
| BBa_K4586003 | ZF21.16 minCMV promoter | This promoter is designed to contain multiple repeats recognized by the zinc finger protein 21.16 to direct VP64 activity toward this promoter. |
| BBa_K4586004 | Cas12k | This part codes for the type V-K CRISPR-Cas system effector protein Cas12k, that is characterized by a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA or genome editing. |
| BBa_K4586005 | MCP-ADAR2 | This part codes for the catalytic portion of improved ADAR2 (adenosine deaminases acting on double-stranded RNA). |
| BBa_K4586006 | Guide RNA | Our guide RNA part is a specific RNA fragment that navigates the Cas system to the RNA or DNA target of interest. |
| BBa_K4586007 | Sensor of the DART V ADAR switch | This part is our novel sensor, which is a single-stranded RNA that recognizes the specific complementary part and initiates the downstream transcription process to activate the CRISPR-Cas system. |
| BBa_K4586008 | MS2 | This part is a highly specific RNA hairpin loop structure that has high affinity for MCP to navigate the MCP-ADAR2 to the sequence of interest by flanking the sequence with two MS2 hairpin structures. |
| BBa_K4586009 | lamp2b | This part is the gene coding for a member of a family of membrane glycoproteins called Lysosome-associated membrane protein 2 (LAMP2), through modification of the outer membrane domain of the lamp2b. Our target protein can be delivered to the exosome's surface using Lamp 2b. |
| BBa_K4586010 | SDC4 | This part is an activator of signaling pathways to control cellular processes that arbitrate cell migration, proliferation, endocytosis, and mechano-transduction, as well as its ability to independently activate signaling pathways in response to the target-ligand binding as well. |
| BBa_K4586011 | STEAP3 | STEAP3 is a metallic reductase enzyme that is also known as TSAP6, which is involved in promoting apoptosis and exosome biogenesis and can reduce iron and copper. It has the ability to convert iron from an insoluble ferric to a soluble ferrous form. |
| BBa_K4586012 | NadB | NadB is the first enzyme in the NAD biosynthesis pathway that is useful in the production of ATP by increasing the metabolism rate of gene-modified cells through the FAD-dependent oxidation of L-aspartate to iminoaspartate, which has an essential role in exosome synthesis. |
| BBa_K4586013 | CX43 | This part is a transmembrane protein called connexin (CX43) that oligomerizes to form hexameric channels, called hemichannels, that can control the interaction and transfer of information between exosomes and cells of interest, which is called intercellular communication. |
| BBa_K4586014 | CD63 | This part is the cell surface protein, which is a member of the tetraspanin family. It has numerous functions, such as being a marker for multivesicular bodies and quantifying extracellular vesicles. |
| BBa_K4586015 | L7Ae | L7Ae is a 50S ribosomal protein that is considered an RNA-binding protein. It has the ability to recognize its K-turn motif or C/D box in ribosomal RNA and can be used to build more complicated genetic circuits. |
| BBa_K4586016 | CMV | CMV promoter is derived from human Cytomegalovirus, which belongs to the Herpesvirus group. All family members share the ability to remain in the latent stage in the human body. CMV is located upstream of the immediate-early gene. |
| BBa_K4586017 | Human U6 promoter | It is a type III RNA polymerase III promoter that controls the expression of short RNAs. Distance sequence element (DSE), proximal sequence element (PSE), and TATA box are the three primary components of the U6 promoter. The nucleotide G indicates the U6 promoter's transcriptional beginning location. |
| BBa_K4586018 | CD 8 alpha signal | CD8 alpha is a signal peptide that translocates the conjugated protein to the cellular membrane. |
| BBa_K4586019 | G4S linker | This part codes for the poly-Glycine-Serine (G4S) linker peptide, that is a type of flexible, unstructured synthetic peptide linker sequence. |
| BBa_K4586020 | SV40 NLS | Nuclear localization signals are short peptides that are responsible for the transport of nuclear protein from the cytoplasm to the nucleus to perform its function. |
| BBa_K4586021 | VP64 | This part codes for VP64, which is a transcriptional activator peptide formed of two components, first, four tandem repeats of VP16 (Herpes simplex viral protein); second, amino acids 437-447. |
| BBa_K4586023 | C\D Box | C\D Box, or kink turn, is a single-stranded RNA that forms a 3D hairpin structure and has an affinity for L7Ae. Therefore, C\D box and L7Ae are used in multiple biological circuits due to their ability to perform variable functions. |
| BBa_K4586030 | Citrullinated vimentin | It is an intermediate filament protein that is citrullinated by peptidyl arginine deiminase 2 (PAD2). |
| BBa_K4586032 | Anti CD19 | This part codes for the light and heavy chain scFv of anti CD19, that represent the extra cellular domain in syn notch receptor sensitive for CD19. |
Characterized Parts
| Characterized (old) Part ID | Our Part ID | Part Name | Type of Characterization |
|---|---|---|---|
| BBa_K2549006 | BBa_K4586001 | Mouse notch core | Literature characterization, Mutational landscape, mathematical modeling, and experimental characterization. |
| BBa_K2549011 | BBa_K4586002 | ZF21.16 | Literature characterization, mathematical modeling, and experimental characterization. |
| BBa_K2619001 | BBa_K4586010 | SDC4 | Literature characterization, Mutational landscape, mathematical modeling, and experimental characterization. |
| BBa_K2796001 | BBa_K4586011 | STEAP3 | Literature characterization, Mutational landscape, mathematical modeling, and experimental characterization. |
| BBa_K2796002 | BBa_K4586012 | NadB | Literature characterization, Mutational landscape, mathematical modeling, and experimental characterization. |
| BBa_K3113072 | BBa_K4586014 | CD63 | Literature characterization, Mutational landscape, and experimental characterization. |
| BBa_K3113009 | BBa_K4586015 | L7Ae | Literature characterization, Mutational landscape, and experimental characterization. |
| BBa_K1119006 | BBa_K4586016 | CMV | Literature characterization, and mathematical modeling. |
| BBa_K2549044 | BBa_K4586018 | CD 8 alpha signal | Literature characterization , and experimental characterization. |
| BBa_K2549053 | BBa_K4586019 | G4S linker | Literature characterization |
| BBa_K2549054 | BBa_K4586020 | SV40 NLS | Literature characterization, mathematical modeling. |
| BBa_J176013 | BBa_K4586021 | VP64 | Literature characterization, mathematical modeling, and experimental characterization. |
| BBa_K3113007 | BBa_K4586023 | C\D Box | Literature characterization |
New Composite Parts
| Part ID | Name | Usage |
|---|---|---|
| BBa_K4586022 | ZF21.16-VP64 | This part is coding for our intracellular transcription module, which is formed of 3 elements. |
| BBa_K4586024 | Synthetic notch(CD8 alpha-his tag-CCP1-mouse notch core-ZF21.16\VP64) | Our design places this receptor on the surface of MSC with a CD8 alpha signal, and it is activated when CCP1 in the extracellular domain connects to the B-cell receptor (BCR) of the autoreactive B cells that synthesize ACPA. |
| BBa_K4586025 | Cargo (guide RNA - switch- Cas12k- MCP/ADAR) | The first component of Cargo is the CRISPR system, where we use guide RNA to direct our Cas12k toward the BAFF-R gene in auto-reactive B-cells, and the second component is the DART V ADAR switch. |
| BBa_K4586026 | Loading system(CD63-L7Ae) | Our loading system is composed of two components: L7Ae, which is coupled to CD63, which is highly expressed on exosomes. Our cargo contains a hairpin structure called a C-D box, and when that structure binds to L7Ae, our cargo is loaded into exosomes. |
| BBa_K4586028 | Exosomal receptor (CCP1, Lamp2b) | We produced CCP1 on the exosomal surface coupled to lysosome-and-associated membrane glycoprotein 2b (LAMP2b) to increase the exosomes' affinity for the autoreactive B-cells. |
| BBa_K4586029 | DART-V-ADAR (tissue-specific switch) | This composite part codes for our novel, modular, and programmable tissue-specific switch based on adenosine deaminase acting on double stranded RNA. |
| BBa_K4586031 | ZF21.16 minCMV promoter - Cre recombinase | This composite part is a translational unit implemented in our project for conditional expression of Cre recombinase enzyme following stimulation of vimentin syn notch receptor. This enzyme controls the conditional expression of our booster genes (SDC4, STEAP3, NadB) (BBa_K4586027) by deleting (STOP) sequence flanked by two loxP sites upstream to the coding of our booster genes. |
| BBa_K4586033 | Exosomal receptor (Anti CD19, Lamp2b) | We expressed Anti CD19 on the exosomal surface coupled to lysosome-and-associated membrane glycoprotein 2b (LAMP2b) to increase the exosomes' affinity for B-cells. |
| BBa_K4586034 | Synthetic notch(CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64) | Our design places this receptor on the surface of MSC with a CD8 alpha signal, and it is activated when Anti CD19 in the extracellular domain binds to CD19 that is highly presented on B-cell membrane. |
New Basic Parts
| Part ID | Name | Description |
|---|---|---|
| BBa_K4586000 | Citrullinated vimentin | It is an intermediate filament protein that is citrullinated by peptidyl arginine deiminase 2 (PAD2). |
| BBa_K4586003 | ZF21.16 minCMV promoter | This promoter is designed to contain multiple repeats recognized by the zinc finger protein 21.16 to direct VP64 activity toward this promoter. |
| BBa_K4586004 | Cas12k | This part codes for the type V-K CRISPR-Cas system effector protein Cas12k, which is characterized by a naturally inactivated RuvC domain and associated with a Tn7-like transposon for RNA-guided DNA or genome editing. |
| BBa_K4586006 | Guide RNA | This part codes for single-stranded RNA that is designed to drive Cas12K activity toward the B-cell activating factor receptor (BAFF-R) gene. |
| BBa_K4586007 | Sensor | This part is a single-stranded RNA and is considered the modular part in our tissue-specific DART V ADAR switch, according to the target tissue or cells. |
| BBa_K4586008 | MS2 | This part codes for the RNA binding sequence that is used in specific techniques that could be performed in multiple applications, such as in the biochemical purification of RNA protein complexes and conjugated to GFP for measuring specific RNA levels in living cells. |
| BBa_K4586009 | LAMP2b | This part is the gene coding for a member of a family of membrane glycoproteins called Lysosome-associated membrane protein 2 (LAMP2). |
| BBa_K4586014 | CX43 | It is a transmembrane protein called connexin (CX43) that facilitates intracellular communication. |
| BBa_K4586017 | U6-promoter | It is a type III RNA polymerase III promoter that controls the expression of short RNAs. |
Improved Parts
| Improved (old) Part ID | Our Part ID | Part Name | Improvement |
|---|---|---|---|
| BBa_K2818001 | BBa_K4586005 | MCP-ADAR2 | MCP-ADAR2 is an improved version of mutant form of ADAR2 enzyme (BBa_K2818001) that was designed by (iGEM18_NTU-Singapore) We conjugated the mutant form ADAR2dd to MS2 coat protein MCP, which specifically binds to short MS2 RNA hairpin 3D structure that replaces the promiscuous dsRNA-interacting domain of natural ADAR. Integrating MCP-ADAR and flanking the sensor UAG codon with two MS2 hairpins allows us to amplify the initial signal of our therapeutic cargo and replace the low background editing of natural ADAR by the targeted and efficient MCP-ADAR2 which ensures maximum activity of our therapeutic cargo within the target auto-reactive B-cells. |
| BBa_K2796028 | BBa_K4586027 | Booster genes (SDC4, STEAP3, NadB) | This composite part is an improvement for (BBa_K2796028) Designed by (iGEM18_LZU-CHINA).We developed this new approach to control the expression of these booster genes (SDC4, STEAP3, NadB) that enhance the exosomes secretion within our engineered mesenchymal stem cell, as we found out that constitutive overexpression of these genes may carry out multiple side effect in addition to depleting MSCs resources and impairing its function and survival, so we implemented the Cre loxP system for conditional gene expression to induce the expression of these booster genes by deleting (STOP) sequence that prevents the transcription of the downstream genes. Thus, the expression of booster genes as condition in the presence of Cre recombinase enzyme CCP1 syn notch activation. |